Method for producing nucleotide by fermentation

ABSTRACT

Nucleoside 5&#39;-phosphate ester is produced by culturing a bacterium belonging to the genus Escherichia having an ability to produce nucleoside 5&#39;-phosphate ester, in which ushA gene and aphA gene do not function normally, in a medium to produce and accumulate nucleoside 5&#39;-phosphate ester in the medium, and collecting the nucleoside 5&#39;-phosphate ester from the medium.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a method for producing nucleotides byfermentation. Nucleotides such as nucleoside 5′-phosphate esters areuseful as seasonings, drugs, raw materials thereof and so forth.

2. Description of the Related Art

As methods for industrial production of nucleoside 5′-phosphate esters,there are known methods comprising producing nucleoside by fermentationand enzymatically phosphorylating the obtained nucleoside to obtainnucleoside 5′-phosphate ester.

On the other hand, methods of directly producing nucleoside 5′-phosphateesters by fermentation have also been proposed. For example, JapanesePatent Publication (Kokoku) No. 56-12438 discloses a method forproducing 5′-guanylic acid, which comprises culturing a mutant strain ofa bacterium belonging to the genus Bacillus showing adenine auxotrophyand resistance to decoyinine or methionine sulfoxide and having anability to produce 5′-guanylic acid (guanosine 5′-monophosphate, alsoabbreviated as “GMP” hereinafter) and collecting GMP produced andaccumulated in the medium. Further, there are several reports onderiving strains which produce 5′-inosinic acid (inosine5′-monophosphate, also abbreviated as “IMP” hereinafter) from inosineproducing strains of Bacillus subtilis (Magasanik, B. et al., J. Biol.Chem., 226, 339 (1957); Fujimoto, M., et al., Agr. Biol. Chem., 30, 605(1966)). However, the production of nucleoside 5′-phosphate esters bydirect fermentation generally suffers from insufficient yield, and it isnot so practical compared with the aforementioned enzymatic methods.

As the reasons for the difficulty of IMP production by directfermentation, there are mentioned bad cell permeability of IMP and quiteubiquitous distribution of degradative enzymes that decompose IMP(Nucleic Acid Fermentation, Edited by Aminosan Kakusan Shudankai,Kodansha Scientific, Japan). To overcome these obstacles, there has beenattempted to delete nucleotide degradative activity. As degradativeenzymes that decompose IMP into inosine, 5′-nucleotidase, acidphosphatase, alkaline phosphatase and so forth are conceived (NucleicAcid Fermentation, supra). Further, the aforementioned Japanese PatentPublication No. 56-12438 also suggests that a bacterial strain showinghigh GMP yield can be obtained from a mutant strain showing reducednucleotidase activity.

As a technique for producing nucleoside 5′-phosphate ester on anindustrial level, a method of producing IMP by using a mutant strain ofBrevibacterium ammoniagenes has been developed (Furuya et al., Appl.Microbiol., 16, 981 (1968)).

As described above, various studies have been made on the production ofnucleoside 5′-phosphate esters by direct fermentation, and somesuccessful examples are also known. However, there are many unknownpoints about nucleotide degradative enzymes, and it cannot be said thatimprovement of yield has been studied sufficiently. In particular, noexample of production of nucleoside 5′-phosphate esters on a practicallevel has been known for bacteria belonging to the genus Escherichia.

SUMMARY OF THE INVENTION

The present invention was accomplished in view of the technicalsituation described above, and an object of the invention is to providea method for producing nucleoside 5′-phosphate ester such as IMP using abacterium belonging to the genus Escherichia.

The inventors of the present invention assiduously studied in order toachieve the aforementioned object. As a result, they found that a genecoding for 5′-nucleotidase other than the known gene existed inEscherichia coli, and successfully identified the gene. Further, theyfound that Escherichia coli having inosine producing ability orguanosine producing ability became to produce IMP or GMP, if the novelgene was disrupted in addition to the known 5′-nucleotidase gene. Thus,they accomplished the present invention.

That is, the present invention provides the followings.

(1) A method for producing nucleoside 5′-phosphate ester, comprising thesteps of culturing a bacterium belonging to the genus Escherichia havingan ability to produce nucleoside 5′-phosphate ester, in which ushA geneand aphA gene do not function normally, in a medium to produce andaccumulate nucleoside 5′-phosphate ester in the medium, and collectingthe nucleoside 5′-phosphate ester from the medium.

(2) The method for producing nucleoside 5′-phosphate ester according to(1), wherein mutations are introduced into the ushA gene and the aphAgene or these genes are disrupted so that they do not function normally.

(3) The method for producing nucleoside 5′-phosphate ester according to(1) or (2), wherein the nucleoside 5′-phosphate ester is selected fromthe group consisting of 5′-inosinic acid or 5′-guanylic acid.

(4) A bacterium belonging to the genus Escherichia having an ability toproduce nucleoside 5′-phosphate ester, in which ushA gene and aphA geneare disrupted.

(5) The bacterium belonging to the genus Escherichia according to (4),wherein the nucleoside 5′-phosphate ester is selected from the groupconsisting of 5′-inosinic acid or 5′-guanylic acid.

(6) A method for searching for a 5′-nucleotidase gene affectingaccumulation of nucleoside 5′-phosphate ester, comprising the steps ofculturing a parent strain of microorganism and a derivative strainthereof in which a known 5′-nucleotidase is deleted in a minimal mediumcontaining a first nucleoside 5′-phosphate ester as a sole carbon sourceand a minimal medium containing a second nucleoside 5′-phosphate esteras a sole carbon source to examine expression profiles of genes in theparent strain and the derivative strain,

calculating a product of a ratio of expression amounts of each gene inthe parent strain and the derivative strain when they are cultured in amedium containing the first nucleoside 5′-phosphate ester as a carbonsource and a ratio of expression amounts of each gene in the parentstrain and the derivative strain when they are cultured in a mediumcontaining the second nucleoside 5′-phosphate ester as a carbon source,and selecting one or more genes showing a larger value of the product.

(7) The method for searching for a 5′-nucleotidase gene according to(6), wherein the first and second nucleoside 5′-phosphate esters are5′-inosinic acid and 5′-guanylic acid.

(8) The method for searching for a 5′-nucleotidase gene according to (6)or (7), further comprising the step of selecting a gene that can codefor a signal sequence required for transition of a protein intoperiplasm from the selected genes.

According to the present invention, nucleoside 5′-phosphate ester suchas IMP and GMP can be produced by direct fermentation using a bacteriumbelonging to the genus Escherichia.

Preferred Embodiments of the Invention

Hereafter, the present invention will be explained in detail.

<1> Search of an Unknown 5′-nucleotidase Gene

As a known 5′-nucleotidase of Escherichia coli, UDP-sugar hydrolase(UshA), which is a product of the ushA gene (GenBank accession X03895),is known. It has been known that the enzyme has 5′-nucleotidase activitythat catalyzes dephosphorylation of nucleoside 5′-phosphate such as AMP,GMP, IMP and XMP to produce a corresponding nucleoside (H. C. Neu,(1967) Journal of Biological Chemistry, 242, 3896-3904; A. Cowman, I. R.Beacham, (1980) Gene, 12, 281-286).

The inventors of the present invention disrupted the ushA gene ofEscherichia coli W3110 strain, and examined its influence on thenucleotide decomposing ability. The 5′-nucleotidase activity inperiplasm of the ushA gene-disrupted W3110 strain (WΔushA) was markedlyreduced compared with the W3110 strain. However, when growth of theWΔushA strain was investigated in a minimal medium containingnucleoside-5′-phosphate as a sole carbon source, this strain could grow.Therefore, it was considered that the nucleotide decomposing ability isnot completely lost by the disruption of only ushA. Furthermore, whennucleoside-5′-phosphate was used as a sole carbon source, start of thegrowth was retarded. Therefore, it was expected that there existedanother 5′-nucleotidase that was induced when UshA did not function.

The inventor of the present invention attempted to search for an unknown5′-nucleotidase gene based on the aforementioned findings, and foundthat a product of a gene reported as an acid phosphatase gene (aphA) (M.C. Thaller, S. Schippa, A. Bonci, S. Cresti, G. M. Rossolini, (1997)FEMS Microbilogy Letters, 146, 191-198, GenBank accession X86971) oryjbP (GenBank accession AAC77025) had the 5′-nucleotidase activity.

A gene coding for such a 5′-nucleotidase that affects the accumulationof nucleoside 5′-phosphate as described above can be searched for asfollows.

First, a microbial parent strain and a derivative strain thereof inwhich a known 5′-nucleotidase is deleted are cultured in a minimalmedium containing a first nucleoside 5′-phosphate ester or a secondnucleoside 5′-phosphate ester such as IMP or GMP as a sole carbonsource. When the microorganism is Escherichia coli, the known5′-nucleotidase may be the aforementioned UshA.

Subsequently, gene expression profiles of these strains areinvestigated. Specifically, a ratio of expression amounts in the wildstrain and the derivative strain is investigated for each gene.

Then, a product of a ratio of expression amounts of a gene in the parentstrain and the derivative strain when they are cultured in a mediumcontaining the first nucleoside 5′-phosphate as a carbon source and aratio of expression amounts of the gene in the parent strain and thederivative strain when they are cultured in a medium containing thesecond nucleoside 5′-phosphate as a carbon source is calculated for eachgene, and one or more genes showing a larger value of the product areselected.

Although the method for gene expression profiling is not particularlylimited, the DNA array method (H. Tao, C. Bausch, C. Richmond, F. R.Blattner, T. Conway, (1999) Journal of Bacteriology, 181, 6425-6440) canbe mentioned, for example.

From the aforementioned selected genes, target genes can be furthernarrowed down by selecting genes that may code a signal sequencerequired for transition of protein to periplasm. This is because it isexpected that the target 5′-nucleotidase transits to periplasm andfunction therein.

As for Escherichia coli, as shown in the examples mentioned later, twokinds of genes, b0220 (also referred to as o157) and yjbP, wereselected. Among these genes, yjbP was an acid phosphatase gene (aphA).On the other hand, b0220 was a gene of which function was unidentified,which was designated as ykfE. When these genes were amplified inEscherichia coli, remarkable increase of 5′-nucleotidase activity wasnot observed in the ykfE gene-amplified strain, whereas remarkableincrease of 5′-nucleotidase activity was observed in the aphAgene-amplified strain. Thus, it was confirmed that the aphA gene product(AphA) had the 5′-nucleotidase activity. In this way, aphA was found asa gene coding for 5′-nucleotidase that affected the accumulation ofnucleoside 5′-phosphate.

<2> Bacterium Belonging to the Genus Escherichia of the PresentInvention

The Bacterium belonging to the genus Escherichia of the presentinvention is a bacterium belonging to the genus Escherichia having anability to produce nucleoside 5′-phosphate, in which the ushA gene andthe aphA gene do not function normally. The Bacterium belonging to thegenus Escherichia itself is not particularly limited so long as it is amicroorganism belonging to the genus Escherichia such as Escherichiacoli. However, specifically, those mentioned in the reference ofNeidhardt et al. (Neidhardt, F. C. et al., Escherichia coli andSalmonella Typhimurium, American society for Microbiology, WashingtonD.C., 1208, Table 1) can be used.

The Bacterium belonging to the genus Escherichia of the presentinvention can be obtained by, for example, breeding a mutant strain orgenetic recombinant strain in which the ushA gene and the aphA gene donot normally function using a Bacterium belonging to the genusEscherichia having purine nucleoside producing ability as a parentstrain. Further, the Bacterium belonging to the genus Escherichia of thepresent invention can also be obtained by breeding similar to thebreeding of purine nucleoside producing strain using a strain in whichthe ushA gene and the aphA gene do not normally function as a parentstrain.

Examples of bacteria belonging to the genus Escherichia having purinenucleoside producing ability include bacteria belonging to the genusEscherichia having an ability to produce inosine, guanosine, adenosine,xanthosine, purine riboside, 6-methoxypurine riboside, 2,6-diaminopurineriboside, 6-fluoropurine riboside, 6-thiopurine riboside,2-amino-6-thiopurine riboside, mercaptoguanosine or the like. Bybreeding a mutant strain or genetic recombinant strain in which the ushAgene and the aphA gene do not normally function using these Escherichiabacteria having purine nucleoside producing ability as a parent strain,bacteria belonging to the genus Escherichia having an ability to producenucleoside 5′-phosphate ester corresponding to each purine nucleosidecan be obtained.

The purine nucleoside producing ability referred to in the presentinvention means an ability to produce and accumulate a purine nucleosidein a medium. Further, the expression of “having purine nucleosideproducing ability” means that the microorganism belonging to the genusEscherichia produces and accumulates a purine nucleoside in a medium inan amount larger than that obtained with a wild strain of E. coli, forexample, the W3110 strain.

Further, the ability to produce nucleoside 5′-phosphate ester means anability to produce and accumulate nucleoside 5′-phosphate ester in amedium. Furthermore, the expression of “having purine nucleosideproducing ability” means that the microorganism belonging to the genusEscherichia produces and accumulates a purine nucleoside in a medium inan amount larger than that obtained with a wild strain of E. coli, forexample, the W3110 strain, and it preferably means that themicroorganism produces and accumulates nucleoside 5′-phosphate ester inan amount of 100 mg/L or more, more preferably 500 mg/L or more, furtherpreferably 1000 mg/L or more, when it is cultured under the conditionsmentioned in Example 6 described later.

Bacteria belonging to the genus Escherichia having purine nucleosideproducing ability are detailed in International Patent PublicationWO99/03988, for example. More specifically, there can be mentioned theEscherichia coli FADRaddG-8-3::KQ strain (purFKQ, purA⁻, deoD⁻, purR⁻,add⁻, gsk⁻) described in the above international patent publication.This strain harbors a mutant purF coding for PRPP amidotransferase ofwhich feedback inhibition by AMP and GMP is desensityzed, and in whichthe lysine residue at a position of 326 is replaced with a glutamineresidue, and a succinyl-AMP synthase gene (purA), purine nucleosidephosphorylase gene (deoD), purine repressor gene (purR), adenosinedeaminase gene (add), and inosine/guanosine kinase gene (gsk) aredisrupted. This strain given with a private number of AJ13334 wasdeposited on Jun. 24, 1997 at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Trade and Industry (currently, the independentadministrative corporation, National Institute of Advanced IndustrialScience and Technology, International Patent Organism Depositary) (ChuoDai-6, 1-1 Higashi 1-Chome, Tsukuba-shi, Ibaraki-ken, Japan, postalcode: 305-5466) as an international deposit under the provisions of theBudapest treaty, and received an accession number of FERM BP-5993. Thisstrain has an ability to produce inosine and guanosine. Further, thestrain obtained by introducing a plasmid containing a mutant purF geneinto the FADRaddeddyicPpgixapA strain, which was constructed asdescribed in the Example to be mentioned later, can also be suitablyused as an inosine producing bacterium. Guanosine producing ability canbe enhanced by introducing the guaA and guaB genes that encode IMPdehydrogenase and GMP synthetase, respectively, into an inosineproducing bacterium. In the present invention, the bacterial strain isnot limited to the aforementioned strains, and any strains having purinenucleoside producing ability can be used without any particularlimitation.

A mutant strain or genetic recombinant strain in which the ushA gene andthe aphA gene do not function normally can be obtained by modifying thegenes so that the activities of 5′-nucleotidases that are the productsof the genes should be decreased or deleted, or transcription of thesegenes should be decreased or eliminated. Such a microorganism can beobtained by, for example, replacing the ushA gene and the aphA gene onthe chromosome with an ushA gene and aphA gene that do not functionnormally (also referred to as “disrupted ushA gene” and “disrupted aphAgene” hereinafter) by homologous recombination utilizing a geneticrecombination method (Experiments in Molecular Genetics, Cold SpringHarbor Laboratory press (1972); Matsuyama, S. and Mizushima, S., J.Bacteriol., 162, 1196 (1985)).

In homologous recombination, a plasmid or the like having a sequenceshowing homology to a sequence on a chromosome is introduced into abacterial cell. Then, recombination occurs at a certain frequency at aposition of the homologous sequence so that the whole introduced plasmidis incorporated into the chromosome. When recombination is furthercaused thereafter at the position of the homologous sequence, theplasmid is again removed from the chromosome. At this time, depending onthe position of the recombination, the disrupted gene may remain on thechromosome, and the original normal gene may be removed together withthe plasmid. By selecting such a bacterial strain, a strain in which thenormal ushA gene or aphA gene on the chromosome is replaced with thedisrupted ushA gene or the disrupted aphA gene can be obtained.

A gene disruption technique based on such homologous recombination hasalready been established, and a method utilizing a linear DNA, a methodutilizing a temperature sensitive plasmid and so forth can be used. Thedisruption of the ushA gene and the aphA gene can also be performed byusing a plasmid containing an ushA gene or aphA gene internally insertedwith a marker gene such as a drug resistance gene, which cannotreplicate in a target microbial cell. That is, in a transformant thatwas transformed with the aforementioned plasmid and hence acquired drugresistance, the marker gene is incorporated into the chromosomal DNA.Since it is highly probable that this marker gene is incorporated intothe chromosome by homologous recombination of the ushA gene or aphA genesequences located on the both ends of the marker gene with those geneson the chromosome, a gene-disrupted strain can be selected efficiently.

The disrupted ushA gene and the disrupted aphA gene used for the genedisruption can be obtained by, specifically, deleting a certain regionof these genes by digestion with a restriction enzyme and ligation,inserting another DNA fragment (marker gene etc.) into these genes, orintroducing substitution, deletion, insertion, addition or inversion ofone or more nucleotides into a nucleotide sequence of coding region,promoter region or the like of the ushA gene or the aphA gene by thesite-specific mutagenesis (Kramer, W. and Frits, H. J., Methods inEnzymology, 154, 350 (1987)) or treatment with a chemical agent such assodium hyposulfite or hydroxylamine (shortie, D. and Nathans, D., Proc.Natl. Acad. Sci. U.S.A., 75, 270 (1978)) so that activity of the encodedrepressor should be decreased or deleted, or transcription of the ushAgene or the aphA gene should be decreased or eliminated. Among theseembodiments, the method of deleting a certain region of the ushA gene oraphA by digestion with a restriction enzyme and ligation and the methodof inserting another DNA fragment into these genes are preferred in viewof certainty and stability of the methods. The order of the genedisruption of the ushA gene and the aphA gene is not particularlylimited, and either one may be disrupted first.

The nucleotide sequences of the ushA gene and the aphA genes themselvesare known, and hence they can be easily obtained by PCR or hybridizationbased on such nucleotide sequences. For example, the ushA gene can beobtained from chromosome DNA of Escherichia coli by PCR using theprimers shown in SEQ ID NOS: 1 and 2, for example. Further, theN-terminal region of the aphA gene can be obtained by PCR using theprimers shown in SEQ ID NOS: 3 and 7, and the C-terminal region of thesame can be obtained by PCR using the primers shown in SEQ ID NOS: 4 and8.

Whether the target gene has been disrupted or not can be confirmed byanalyzing the gene on a chromosome by Southern blotting or PCR.

<3> Method for Producing nucleoside 5′-phosphate ester

Nucleoside 5′-phosphate ester can be produced by culturing a bacteriumbelonging to the genus Escherichia having an ability to producenucleoside 5′-phosphate ester, in which the ushA gene and the aphA genedo not function normally, in a medium to produce and accumulatenucleoside 5′-phosphate ester in the medium, and collecting thenucleoside 5′-phosphate ester from the medium.

The medium may be a usual medium containing a carbon source, nitrogensource, inorganic ions, and other organic components, if needed. As thecarbon source, there can be used saccharides such as glucose, lactose,galactose, fructose, arabinose, maltose, xylose, trehalose, ribose andstarch hydrolysate, alcohols such as glycerol, mannitol and sorbitol,organic acids such as gluconic acid, fumaric acid, citric acid andsuccinic acid and so forth.

As the nitrogen source, there can be used inorganic ammonium salts suchas ammonium sulfate, ammonium chloride, and ammonium phosphate, organicnitrogen such as soybean hydrolysate, ammonia gas, aqueous ammonia andso forth.

As the organic trace nutrients, it is desirable to add requiredsubstances including vitamins such as vitamin B1, nucleic acids such asadenine and RNA or yeast extract in a suitable amount. In addition tothese, a small amount of potassium phosphate, magnesium sulfate, ironions, manganese ions and so forth are added as required.

Culture is preferably carried out under an aerobic condition for 16-72hours. The culture temperature is controlled to be 30° C. to 45° C., andpH is controlled to be 5 to 8 during the culture. Inorganic or organic,acidic or alkaline substances as well as ammonia gas and so forth can beused for pH adjustment.

Collection of nucleoside 5′-phosphate ester from fermented liquor isusually carried out by a combination of an ion exchange resin method, aprecipitation method and other known techniques.

BEST MODE FOR CARRYING OUT THE INVENTION

The present invention will be further specifically explained hereinafterwith reference to the following examples.

EXAMPLE 1 Effect of ushA Disruption on nucleotide Production ofEscherichia coli

<1> Construction of ushA-Disrupted Strain

From genomic DNA of the Escherichia coli W3110 strain, a ushA genefragment was amplified by PCR. The genomic DNA was extracted by usingRNA/DNA maxi Kit (produced by Qiagen). PCR was performed by using theprimers shown in SEQ ID NOS: 1 and 2 and Pyrobest DNA Polymerase(produced by Takara Shuzo) according to the instruction appended to thepolymerase. After PCR, the amplified DNA fragments were purified byusing Wizard PCR Preps (produced by Promega). After digestion withrestriction enzymes SphI and SalI (produced by Takara Shuzo), thepurified DNA fragments were subjected to a phenol/chloroform treatmentand ethanol precipitation. pHSG397 (produced by Takara Shuzo) similarlydigested with SphI and SalI was ligated by using DNA ligation Kit Ver.2(produced by Takara Shuzo). Competent cells of JM109 (produced by TakaraShuzo) were transformed with the above ligation mixture, and plated onan LB agar plate containing 30 μg/mL of chloramphenicol (produced bySigma) (LB+chloramphenicol plate). After culturing at 37° C. overnight,grown colonies were cultured in LB medium containing 30 μg/mL ofchloramphenicol at 37° C. in a test tube, and a plasmid was extractedusing an automatic plasmid extractor, PI-50 (produced by KuraboIndustries). The obtained plasmid was designated as pHSGushA.

Then, an HpaI fragment was removed from the ushA gene contained inpHSGushA as follows. pHSGushA was digested with a restriction enzymeHpaI (produced by Takara Shuzo), subjected to a phenol/chloroformtreatment and ethanol precipitation, and ligated by using DNA LigationKit Ver.2. JM109 was transformed with this ligation solution, and aplasmid was extracted from emerged colonies. The obtained plasmid wasdigested with SphI and SalI, and subjected to agarose gelelectrophoresis to select a plasmid containing an inserted targetfragment in which the HpaI digestion fragment was deleted from the ushAgene region.

The obtained plasmid fragment and a fragment obtained by digesting thetemperature sensitive plasmid pMAN997 described in International PatentPublication WO99/03988 with SphI and SalI were ligated. JM109 wastransformed with the ligation solution, and colonies were selected at30° C. on an LB agar plate containing 50 μg/mL of ampicillin (producedby Meiji Seika Kaisha) (LB+ampicillin plate). The colonies were culturedin LB medium containing 50 μg/mL of ampicillin at 30° C. in a test tube,and plasmids were extracted. A plasmid from which a fragment of adesired length could be obtained by digestion with SphI and SalI wasused as a plasmid for ushA disruption, pMANΔushA. The above pMAN997 wasobtained by exchanging VspI-HindIII fragments of pMAN031 (J. Bacteriol.,162, 1196 (1985)) and pUC19 (produced by Takara Shuzo).

The W3110 strain was transformed with pMANΔushA, and colonies wereselected on an LB+ampicillin plate at 30° C. The selected clones werecultured at 30° C. overnight as liquid culture. The culture broth wasdiluted 10⁻³times, and inoculated on an LB+ampicillin plate, andcolonies were selected at 42° C. The selected clones were applied andspread on an LB+ampicillin plate, and cultured at 30° C. Then, ⅛ of thecells on the plate were suspended in 2 mL of LB medium, and cultured at42° C. for 4 to 5 hours with shaking. The cells diluted 10⁻⁵ times wereseeded on an LB plate, and several hundreds of colonies among theobtained colonies were inoculated on an LB plate and LB+ampicillinplate, and growth was confirmed to select ampicillin sensitive strains.Colony PCR was performed for several strains among the ampicillinsensitive strains to confirm the deletion of ushA gene. In this way, anushA-disrupted strain derived from E. coli W3110, WΔushA, was obtained.

<2> Measurement of 5′-nucleotidase and nucleotide Assimilation Culture

W3110 and WΔushA were cultured at 37° C. in LB medium, and periplasm wasextracted from cells in a proliferation phase according to the method ofEdwards et al. (C. J. Edwards, D. J. Innes, D. M. Burns, I. R. Beacham,(1993) FEMS Microbiology Letters, 114, 293-298). By using the proceduredescribed in the above reference, 5′-nucleotidase activity ofperiplasmic proteins for IMP, GMP and AMP was measured. Activityproducing 1 μmol of phosphoric acid per minute was defined as 1 unit. Asa result, the periplasmic 5′-nucleotidase activity of WΔushA wasmarkedly decreased compared with W3110 as shown in Table 1. TABLE 1Periplasmic 5′-nucleotidase activity (Unit/mg of protein) SubstrateStrain IMP GMP AMP W3110 14.0 10.8 14.2 WΔushA 0.21 0.16 0.03

In order to confirm whether WΔushA had completely lost the nucleotidedecomposition ability, its growth was investigated in a minimal mediumcontaining a nucleotide as a sole carbon source. W3110 and WΔushA werecultured overnight at 37° C. in LB medium, then washed withphysiological saline, added to 50 mL of M9 minimal medium (J. H. Miller,“A SHORT COURSE IN BACTERIAL GENETICS”, Cold Spring Harbor LaboratoryPress, New York, 1992) containing 5.8 g/L of IMP or 6.7 g/L of GMP, andcultured at 37° C. After a suitable time had passed, the culture brothwas collected and its absorbance at 600 nm was measured by using aspectrophotometer DU640 (produced by Beckman). Although the growth ofWΔushA degraded in M9 medium containing IMP or GMP as a carbon source,it could grow in such a medium. This suggested that the nucleotidedegradative ability was not completely lost by the disruption of onlyushA. Further, since the start of growth was retarded, existence ofanother 5′-nucleotidase was expected, which was induced when UshA didnot function.

EXAMPLE 2 Search of Novel 5′-nucleotidase Gene

It was considered that the 5′-nucleotidase gene predicted in Example 1was more strongly expressed in WΔushA compared with W3110 when they werecultured in M9 medium containing IMP or GMP as a carbon source. In orderto identify the 5′-nucleotidase considered to function in WΔushA, geneexpression profiles of W3110 and WΔushA cultured in M9 medium containingIMP or GMP as a carbon source were compared.

For comparison of gene expression profiles, the DNA array method (H.Tao, C. Bausch, C. Richmond, F. R. Blattner, T. Conway, (1999) Journalof Bacteriology, 181, 6425-6440) was used. Panorama E. coli Gene Arrays(produced by Sigma Genosis) is a DNA array composed of a nylon membranespotted with amplified DNA fragment of 4290 genes of E. coli, and mRNAexpression amounts of the total genes of E. coli can be comprehensivelyanalyzed at once by using it.

W3110 and WΔushA were cultured in M9 medium containing IMP or GMP as asole carbon source, and RNA was extracted from the cells at aproliferation phase by using RNeasy mini Kit (produced by Qiagen). Theextracted RNA solution was added with MgCl₂ and DNaseI (BoeringerMannheim) at final concentrations of 10 mM and 0.25 U/ml, respectively,to decompose contaminated genomic DNA, and the total RNA were thenpurified by phenol/chloroform extraction and ethanol precipitation. Areverse transcription reaction was performed by using AMV reversetranscriptase (produced by Promega), dATP, dGTP, dTTP, [α-³³P]-dCTP (allproduced by Amersham Pharmacia), and random primer pd(N)₆ (produced byAmersham Pharmacia) according to the instructions appended to PanoramaE. coli Gene Arrays to prepare a cDNA probe. The obtained cDNA probe waspurified by using ProbeQuant (produced by Amersham Pharmacia).

By using the cDNA probe obtained above, hybridization and washing wereperformed according to the instruction appended to Panorama E. coli GeneArrays. The membrane was enclosed in a hybridization bag, and broughtinto contact with an imaging plate (produced by Fuji Photo Film) for 48hours, and an image was captured by using FLA3000G (produced by FujiPhoto Film). Concentration of each spot was quantified by using imageanalysis software, AIS (produced by Imaging Research), and ratio of eachspot concentration with respect to the sum of the total spotconcentrations on the same membrane was represented for every membrane.Increase and decrease of gene expression was investigated by comparingvalues of this ratio for each gene.

In this way, genes of which expression amount were larger in WΔushAcompared with W3110 when they were cultured in M9 medium containing IMPas a carbon source, and genes of which expression amount were larger inWΔushA compared with W3110 when they were cultured in M9 mediumcontaining GMP as a carbon source were selected, respectively. However,since the change of the carbon source for the culture might causevariation of expression amounts of many genes, the number of selectedgenes was large, and it was difficult to confirm function of each gene.Therefore, as means for narrowing down the candidate genes, thefollowing screening method was employed.

Since it was considered that the target 5′-nucleotidase gene showedincreased expression amount in both of the cultures utilizing IMP andGMP as the carbon source, a product of a ratio of expression amounts inWΔushA and W3110 (WΔushA/W3110) obtained when they were cultured withIMP as the carbon source and a ratio of expression amounts in WΔushA andW3110 (WΔushA/W3110) obtained when they were cultured with GMP as thecarbon source was calculated, and a gene showing a large value for theproduct was searched for. The genes that showed larger values of top 50are shown in Table 2 (1-25th places) and Table 3 (26-50th places). Amongthese, genes of which functions were unknown were selected as candidatesthat might have the 5′-nucleotidase activity. Since WΔushA could grow bydecomposing extracellular nucleotides, it was expected that the target5′-nucleotidase should migrate to periplasm and function therein.Therefore, from those genes of which functions were unknown, only thosehaving a signal sequence required for transition of protein to periplasmwere selected. By these screenings, the candidate genes were narroweddown to two kinds, b0220 (or o157) and yjbP.

When these genes were investigated, it was found that b0220 was a genereported as a gene of unidentified function designated as ykfE, and yjbPwas a gene reported as an acid phosphatase gene (aphA) (M. C. Thaller,S. Schippa, A. Bonci, S. Cresti, G. M. Rossolini, (1997) FEMSMicorobilogy Letters, 146, 191-198). TABLE 2 Gene expression profilesobserved in W3110 and WΔushA when they were cultured in M9 mediumcontaining IMP or GMP as carbon source (1-25th places) IMP expressionGMP expression Ratio (I) ratio (G) I × G Gene 11.3 5.5 61.7 pyrB 3.5 7.325.2 malE 4.5 2.0 9.1 pyrI 3.6 2.2 8.0 udp 3.9 2.0 7.9 deoD 2.8 2.6 7.2yeiN 1.9 3.7 7.2 lamB 5.1 1.2 6.0 b0220 (o157) 3.5 1.7 5.9 DeoA 2.1 2.75.5 YeiC 2.1 2.6 5.4 tsx 3.0 1.8 5.3 b1036 (o173) 4.2 1.2 4.9 DeoC 2.32.1 4.8 NupC 2.4 2.0 4.8 FadB 2.1 2.3 4.8 YejD 1.5 3.2 4.8 MalF 1.9 2.34.4 CirA 2.6 1.7 4.3 CarA 1.5 2.9 4.2 LivJ 3.2 1.3 4.0 TalB 0.9 4.5 4.0FliD 1.5 2.6 4.0 MalM 1.6 2.4 3.9 DppA 1.0 4.0 3.8 FliC

TABLE 3 Gene expression profiles observed in W3110 and WΔushA when theywere cultured in M9 medium containing IMP or GMP as carbon source(26-50th places) IMP expression GMP expression Ratio (I) Ratio (G) I × GGene 0.8 4.4 3.7 CheA 2.8 1.3 3.7 DeoB 1.3 2.7 3.6 GlpK 2.1 1.7 3.5b2341 (f714) 1.8 1.8 3.3 YeiK 2.8 1.2 3.3 Cdd 2.0 1.6 3.2 b2673 (o81)1.8 1.7 3.1 YeiP 1.9 1.7 3.1 YeiR 0.9 3.3 3.0 MotB 3.1 1.0 3.0 YafP 2.01.5 3.0 bO221 (f826) 1.6 1.8 2.9 yjbP 0.7 4.0 2.9 tap 1.9 1.5 2.9 pyrH1.5 1.9 2.8 sseA 1.8 1.6 2.8 ybeK 0.8 3.3 2.7 flgN 1.9 1.4 2.7 glnA 2.01.3 2.7 ygaD 2.3 1.2 2.7 entE 1.7 1.6 2.6 yafY 1.9 1.4 2.6 nupG 1.8 1.72.6 fepA 1.2 2.2 2.6 b3524 (hypothetical)

EXAMPLE 3 Evaluation of Candidate Genes by Gene Amplification

Strains in which the candidate genes obtained in Example 2, ykfE andaphA, were each amplified were prepared to investigate the influence ofthe gene amplification on the 5′-nucleotidase activity. The genefragments of ykfE and aphA were amplified by using the primers shown inSEQ ID NOS: 3 and 4, and the primers shown in SEQ ID NOS: 5 and 6,respectively. The ykfE fragment was cloned into a vector pSTV28(produced by Takara Shuzo) at a cleavage site obtained with restrictionenzymes SalI and PstI (produced by Takara Shuzo) to obtain pSTVykfE.Further, the aphA fragment was cloned into pSTV28 at a cleavage siteobtained with SalI and SphI to obtain pSTVaphA. WΔushA was transformedwith each of the plasmids prepared as described above, and cultured at37° C. in LB medium containing 30 μg/mL of chloramphenicol. The5′-nucleotidase activity for IMP, GMP and AMP as a substrate inperiplasm of cells in a proliferation phase was measured. As a result,the aphA gene amplification provided marked increase of the5′-nucleotidase activity compared with a strain harboring only thevector as shown in Table 4, and thus it was confirmed that the AphAprotein had the activity. On the other hand, the ykfE-amplified straindid not show significant increase of the activity, and thus it wasdetermined that it did not have the 5′-nucleotidase activity. TABLE 45′-Nucleotidase activity in periplasm of aphA- and ykfE-amplifiedstrains (U/mg of protein) Substrate Strain IMP GMP AMP WΔushA/pSTV 0.0740.067 0.024 WΔushA/pSTVykfE 0.15 0.15 0.067 WΔushA/pSTVaphA 3.2 3.5 1.8

EXAMPLE 4 Introduction of aphA Disruption into WΔushA

Gene disruption was performed in WΔushA strain for aphA, which wasexpected to be a gene for the 5′-nucleotidase activity. A fragment ofthe N-terminus region and fragment of the C-terminus region of aphA wereamplified by PCR using the primers shown in SEQ ID NOS: 3 and 7 and theprimers shown in SEQ ID NOS: 4 and 8, respectively, and purified byusing Wizard PCR Preps. The amplification reaction solutions in anamount of 1 μL each were mixed, added to a PCR reaction solution andsubjected to crossover PCR (A. J. Link, D. Phillips, G. M. Church (1997)Journal of Bacteriology, 179, 6228-6237) using the primers shown in SEQID NOS: 3 and 4 to obtain an aphA gene fragment including deletion ofits center portion of about 300 nucleotides. This fragment was insertedinto an SalI-SphI cleavage site of temperature sensitive plasmid pMAN997to obtain a plasmid pMANΔaphA for gene disruption. By using this plasmidfor gene disruption, each aphA of W3110 and WΔushA was disrupted toobtain an aphA-deficient strain (WΔaphA) and ushA- and aphA-doubledeficient strain (WΔushAΔaphA).

EXAMPLE 5 Measurement of 5′-nucleotidase Activity and nucleotideAssimilation Culture of WΔushAΔaphA

W3110, WΔushA, WΔaphA and WΔushAΔaphA were each cultured at 37° C. in LBmedium, and 5′-nucleotidase activity in periplasm of cells in aproliferation phase was measured. The results are shown in Table 5.Although the activity in WΔaphA was reduced about by half compared withW3110, it still strongly remained, and it was considered that ushAcontributed to it. On the other hand, the 5′-nucleotidase activity inthe periplasm of WΔushAΔaphA, which was a double-deficient strain, wasfurther reduced and substantially eliminated. TABLE 5 5′-Nucleotidaseactivity of W3110, WΔushA, WΔaphA, and WΔushAΔaphA (U/mg of protein)Substrate Strain IMP GMP AMP XMP W3110 14.0 10.9 14.2 8.7 WΔaphA 5.8 4.16.0 3.9 WΔushA 0.21 0.16 0.03 0.10 WΔushAΔaphA 0.010 0.009 0.012 0.019

Furthermore, in order to investigate the nucleotide degradative abilityof each strain, these strains were cultured in M9 medium containing IMPor GMP as a carbon source in flasks. While growth was observed forW3110, WΔaphA and WΔushA with both of the carbon sources with growthintensities in that order, growth was not observed for WΔushAΔaphA eventhough it was cultured for 300 hours, and thus it was revealed that itcould not grow in M9 medium containing IMP or GMP as a sole carbonsource. In this way, the ability to decompose extracellular nucleotideof E. coli W3110 was successfully deleted by double deficiency of ushAand aphA.

EXAMPLE 6 Gene Disruption for ushA and aphA in Inosine ProducingBacterium

In order to investigate the possibility of direct fermentation of IMP,the gene disruption was performed for ushA and aphA in an inosineproducing strain of Escherichia coli. As the inosine producingbacterium, FADRaddeddyicPpgixapA (referred to as “I” hereinafter)described in International Patent Publication WO99/03988 was used. Themutant purF gene fragment contained in the plasmid pKFpurFKQ mentionedin WO99/03988 was digested with BamHI and HindIII, then purified andligated to pMW218 (produced by Nippon Gene) digested with the sameenzymes. The obtained plasmid pMWpurFKQ was introduced into the Istrain. The obtained strain, I/pMWpurFKQ, became a strain having abilityto accumulate about 2-3 g/L of inosine in culture broth.

The aforementioned strain FADRaddeddyicPpgixapA was a strain in whichPRPP amidotransferase gene (purF), succinyl-AMP synthase gene (purA),purine nucleoside phosphorylase gene (deoD), purine repressor gene(purR), adenosine deaminase gene (add), 6-phosphogluconate dehydrasegene (edd), adenine deaminase gene (yicP), phosphoglucose isomerase gene(pgi) and xanthosine phosphorylase gene (xapA) were disrupted. Further,pKFpurFKQ contained a mutant purF coding for PRPP amidotransferase inwhich the 326th lysine residue was replaced with a glutamine residue,and of which feedback inhibition by AMP and GMP was canceled (seeInternational Patent Publication WO99/03988).

By using the aforementioned plasmid pMANΔushA for ushA gene disruptionand the plasmid pMANΔaphA for aphA gene disruption, a ushA-singledeficient strain (IΔushA/pMWpurFKQ), aphA-single deficient strain(IΔaphA/pMWpurFKQ) and ushA- and aphA-double deficient strain(IΔushAΔaphA/pMWpurFKQ) were obtained.

Each of the aforementioned strains was evaluated for IMP producingability. Medium, culture methods and analysis method for the evaluationof IMP producing ability are shown below.

[Base Medium: MS Medium] Final concentration Glucose 40 g/L (separatelysterilized) (NH₄)₂SO₄ 16 g/L KH₂PO₄ 1 g/L MgSO₄7H₂O 1 g/L FeSO₄7H₂O 0.01g/L MnSO₄4H₂O 0.01 g/L Yeast extract 8 g/L CaCO₃ 30 g/L (separatelysterilized)[Culture Method]

Refresh culture: stored cells were inoculated, LB agar medium (addedwith necessary agents), 37° C., overnight.

Seed culture: refreshed cells were inoculated, LB broth (added withnecessary agents), 37° C., overnight.

Main culture: seed culture broth was inoculated in an amount of 2%, MSmedium (added with adenine and other agents as required), 37° C., 20 ml,in 500-ml volume Sakaguchi flask.

[Analysis Method]

In an amount of 500 μl of the culture broth was sampled in a timecourse, and centrifuged at 15,000 rpm for 5 minutes, and the supernatantwas diluted 4 times with H₂O and analyzed by HPLC.

Analysis Conditions: Column: Asahipak GS-220 (7.6 mm ID × 500 mm L)Buffer: 0.2 M NaH₄PO₄ (adjusted to pH 3.98 with phosphoric acid)Temperature: 55° C. Flow rate: 1.5 ml/min Detection: UV 254 nm Retentiontime (min) Inosine 16.40 IMP 11.50 Guanosine 19.67 GMP 13.04

The results are shown in Table 6. In Table 6, results of two parallelexperiments are indicated, respectively. It was demonstrated thatIΔushAΔaphA accumulated about 1.0 g/L at most of IMP in the culturebroth. TABLE 6 Evaluation of ushA- and aphA-deficient strains of inosineproducing bacterium by culture in flask Culture time Inosine IMP Strain(h) (g/L) (g/L) I/pMWpurFKQ 48 2.3 0 48 2.3 0 IΔushA/pMWpurFKQ 51 3.1 051 2.9 0 IΔaphA/pMWpurFKQ 51 3.6 0 51 3.2 0 IΔushAΔaphA/pMWpurFKQ 54 2.41.0 54 2.6 0.6

EXAMPLE 7 Production of GMP by ushA- and aphA-Double Deficient Strain

In order to examine the possibility of GMP production by the presentinvention, guanosine producing ability was imparted to the ushA- andaphA-double deficient strain obtained in Example 6,IΔushAΔaphA/pMWpurFKQ. Impartation or enhancement of guanosine producingability was attained by enhancing genes of enzymes catalyzing reactionsfrom IMP to GMP. The reaction converting IMP to XMP is catalyzed by IMPdehydrogenase encoded by guaA, and the reaction converting XMP to GMP iscatalyzed by GMP synthetase encoded by guaB, and it is known that thesegenes constitute an operon (guaBA) in Escherichia coli. Therefore, PCRwas performed by using the primer shown in SEQ ID NOS: 9 and 10 toamplify guaBA operon of Escherichia coli. The amplified fragment waspurified, and the restriction enzyme sites formed on the both ends weredigested with SacI and KpnI. The digested fragment was ligated to pSTV28similarly digested with SacI and KpnI, and a plasmid pSTVguaBA intowhich the guaBA gene was incorporated was selected. This plasmid cancoexist with the plasmid pMWpurFKQ harbored by IΔushAΔaphA/pMWpurFKQ.

The aforementioned pSTVguaBA was introduced into theIΔushAΔaphA/pMWpurFKQ strain to obtain IΔushAΔaphA/pMWpurFKQ/pSTVguaBAstrain. Further, as a control, IΔushAΔaphA/pMWpurFKQ/pSTV28 strain wasprepared, which was introduced with the vector pSTV28.

According to the same culture methods and analysis method as in Example6, inosine, IMP, guanosine and GMP accumulated in the culture broth werequantified for the IΔushAΔaphA/pMWpurFKQ/pSTVguaBA strain andIΔushAΔaphA/pMWpurFKQ/pSTV28 strain. The results are shown in Table 7.In the IΔushAΔaphA/pMWpurFKQ/pSTV28 strain used as a control, theculture time was prolonged due to the influence of the introduction ofpSTV28, and it provided a result different from that of theIΔushΔaphA/pMWpurFKQ/pSTVguaBA strain. Guanosine could not bequantified, since its peaks overlapped with other peaks. On the otherhand, it was demonstrated that the IΔushAΔaphA/pMWpurFKQ/pSTVguaBAstrain accumulated about 0.1 g/L of GMP in the culture broth thanks tothe introduction of guaBA. TABLE 7 Culture of ushA- and aphA-deficientstrain of inosine producing bacteria in flask Culture Inosine IMPGuanosine GMP Strain time (h) (g/L) (g/L) (g/L) (g/L) IΔushAΔaphA/ 789.7 0.4 —* 0.0 pMWpurFKQ/pSTV28 IΔushAΔaphA/ 78 3.4 0.2 1.1 0.1PMWpurFKQ/PSTVguaBA*indicates that quantification was not possible.

1-8. (canceled)
 9. A method for producing nucleoside 5′-phosphate ester,comprising the steps of culturing a bacterium belonging to Escherichiacoli having an ability to produce nucleoside 5′-phosphate ester, inwhich expression of ushA gene and aphA gene is decreased as compared toa wild type strain by mutating or disrupting the ushA gene and the alphagene, in a medium containing glucose or sucrose to produce andaccumulate nucleoside 5′-phosphate ester in a medium by directfermentation, and collecting the nucleoside 5′-phosphate ester isselected from the group consisting of inosine 5′-phosphate ester andguanosine 5′-phosphate ester.
 10. The method according to claim 9,wherein the bacterium is further transformed with the mutant purF geneof Escherichia coli coding for PRPP amidotransferase in which the lysineresidue at position 326 is replaced with a glutamine residue.
 11. Themethod according to claim 10, wherein the bacterium is furthertransformed with a guaBA operon of Escherichia coli.
 12. The methodaccording to claim 9, wherein the nucleoside 5′-phosphate ester isinosine 5′-phosphate ester.
 13. The method according to claim 9, whereinthe nucleoside 5′-phosphate ester is guanosine 5′-phosphate ester.